The long-term goal of this research is to understand the molecular mechanisms that lead to the emergence of pandemic influenza A viruses. This knowledge is critical to promoting an increasing preparedness for global influenza outbreaks such as the 'Spanish Influenza' of 1918/1919. Recent direct transmission of avian influenza A viruses to Hong Kong residents has increased concern over the generation of a new pandemic strain. An added concern is the potential use of influenza viruses as bioterrorism agents. The proposed research takes advantage of a plasmid-based reverse genetics system, developed in the applicant's laboratory, to generate influenza viruses entirely from cloned cDNA. Aim 1 seeks to define the contributions of the 1918 influenza virus HA and NA genes to the unprecedented virulence of this strain, focusing on receptor specificity/affinity and fusion activity of the HA and on the sialidase activity of the NA. Aim 2 investigates the cellular and molecular basis of life-threatening lung hemorrhage associated with a 1918-like recombinant virus in mice, testing a multifactorial hypothesis that involves cytokine expression levels, neutrophil-mediated injury, and coagulation defects, all stemming from viral infection. Aim 3 tests complementary hypotheses that would explain the generation of new pandemic virus lineages and viral adaptation to human host cells. That is, 'mutator mutants' (viruses with error-prone polymerase complexes) are predicted to generate greater-than-normal genetic variability in the viral population, yielding mutants optimally adapted to their host. This concept will be evaluated by comparing the mutation rates of isolates that founded new lineages with those of their descendent viruses. Second, host environments conducive to adaptive changes may be required before wild waterfowl viruses can be transmitted to humans. The role of land-based poultry as an intermediate host will be tested by passaging viruses, isolated from waterfowl, in chickens or quail and then determining any adaptive changes in HA receptor binding and NA sialidase activity, both of which are critical determinants of host range restriction. Collectively, these studies will help to elucidate the mechanism(s) underlying the emergence of new (pandemic) influenza A virus lineages, a crucial step in combating future threats from this virus. [unreadable] [unreadable] [unreadable]